|Year : 2016 | Volume
| Issue : 6 | Page : 748-755
Influence of angiotensin converting enzyme (ACE) gene rs4362 polymorphism on the progression of kidney failure in patients with autosomal dominant polycystic kidney disease (ADPKD)
Gnanasambandan Ramanathan1, Santu Ghosh2, Ramprasad Elumalai3, Soundararajan Periyasamy3, Bhaskar V.K.S. Lakkakula1
1 Department of Biomedical Sciences, Sri Ramachandra University, Chennai, India
2 Department of Environmental Health Engineering, Sri Ramachandra University, Chennai, India
3 Department of Nephrology, Sri Ramachandra University, Chennai, India
|Date of Submission||09-Dec-2013|
|Date of Web Publication||12-Oct-2016|
Bhaskar V.K.S. Lakkakula
Sickle Cell Institute, Chattisgarh, Genetic Lab, Department of Biochemistry, Pt. J.N.M. Medical College, Raipur 492 001, Chattisgarh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background & objectives: Autosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disorder, characterized by the fluid filled cysts in the kidneys leading to end stage renal failure in later years of life. Hypertension is one of the major factors independently contributing to the chronic kidney disease (CKD) progression. The renin-angiotensin aldosterone system (RAAS) genes have been extensively studied as hypertension candidate genes. The aim of the present study was to investigate the role of angiotensin converting enzyme tagging - single nucleotide polymorphisms (ACE tag-SNPs) in progression of CKD in patients with ADPKD.
m0 ethods: In the present study six ACE tagSNPs (angiotensin converting enzyme tag single nucleotide polymorphisms) and insertion/deletion (I/D) in 102 ADPKD patients and 106 control subjects were investigated. The tagSNPs were genotyped using FRET-based KASPar method and ACE ID by polymerase chain reaction (PCR) and electrophoresis. Genotypes and haplotypes were compared between ADPKD patients and controls. Univariate and multivariate logistic regression analyses were performed to assess the effect of genotypes and hypertension on CKD advancement. Mantel-Haenszel (M-H) stratified analysis was performed to study the relationship between different CKD stages and hypertension and their interaction.
Results: All loci were polymorphic and except rs4293 SNP the remaining loci followed Hardy-Weinberg equilibrium. Distribution of ACE genotypes and haplotypes in controls and ADPKD patients was not significant. A significant linkage disequilibrium (LD) was observed between SNPs forming two LD blocks. The univariate analysis revealed that the age, hypertension, family history of diabetes and ACE rs4362 contributed to the advancement of CKD.
Interpretation & conclusions: The results suggest that the ACE genotypes are effect modifiers of the relationship between hypertension and CKD advancement among the ADPKD patients.
Keywords: ACE gene - ADPKD - CKD - gene-environment interaction - tagSNP
|How to cite this article:|
Ramanathan G, Ghosh S, Elumalai R, Periyasamy S, Lakkakula BV. Influence of angiotensin converting enzyme (ACE) gene rs4362 polymorphism on the progression of kidney failure in patients with autosomal dominant polycystic kidney disease (ADPKD). Indian J Med Res 2016;143:748-55
|How to cite this URL:|
Ramanathan G, Ghosh S, Elumalai R, Periyasamy S, Lakkakula BV. Influence of angiotensin converting enzyme (ACE) gene rs4362 polymorphism on the progression of kidney failure in patients with autosomal dominant polycystic kidney disease (ADPKD). Indian J Med Res [serial online] 2016 [cited 2019 Sep 15];143:748-55. Available from: http://www.ijmr.org.in/text.asp?2016/143/6/748/191992
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited systemic disorder, characterized by the fluid filled cysts in the kidneys leading to end stage renal failure in later years of life  . Although the exact genetic factors that favour renal progression have not been identified, studies on mouse and human models suggest that the genetic variations play a significant role in the differential renal progression that is observed in ADPKD , . Intra-familial heterogeneity that is found in the expression of ADPKD, suggests the involvement of environmental factors along with the genetic factors in the progression of ADPKD  . Polycystic kidney disease genes products share sequence homology and are thought to play an important role in cell-cell and cell-matrix interactions and in structure of a receptor-channel complex which regulates renal ion transport  . Patients with ADPKD exhibit non-uniformity of the renal disease progression. This variability was observed from both mouse  and human  models, suggesting that disease-modifying loci may play a role in the variable renal progression found in ADPKD.
Hypertension is one of the clinical manifestations in majority of ADPKD cases and correlates with the progressive kidney enlargement with high prognostic values. The renin-angiotensin aldosterone system (RAAS) is known to regulate the blood pressure and fluid balance  . The activity of RAAS system is regulated by the rate of production of angiotensinogen from rennin that is mediated by angiotensin converting enzyme (ACE)  . In view of the role of hypertension on the progression of chronic kidney disease in ADPKD, the gene polymorphisms of ACE are of great interest.
The ACE gene spans over 24 kb with 26 exons and is located on chromosome 17. The presence or absence of a 287-bp repeat sequence [insertion/deletion (I/D) polymorphism] at intron 16 has been used as a common marker in the susceptibility of various disorders  . An initial study showed a deleterious effect of the DD genotype on renal survival in ADPKD  . t0 he direct biological mechanism by which ACE ID polymorphism might influence serum ACE levels remains unclear. In the present study the role of ACE ID and six additional tagging-single nucleotide polymorphisms (tagSNPs) were investigated to unravel the ACE gene modifier effect for renal disease progression in patients with ADPKD.
| Material & Methods|| |
A total of 102 consecutive patients affected by ADPKD (55.88% men) and 106 controls (60.38% men) attending the outpatient department of Nephrology, Sri Ramachandra University, Chennai, India, from March 2010 to December 2012 were consecutively selected. The diagnosis of ADPKD was based on previously described Ravine ultrasound criteria  . Patients with other conditions that could influence renal function such as diabetes or pregnancy were excluded. Upon screening, a total of 106 healthy unrelated individuals without family history of polycystic kidney disease or any other kidney related complications (60.38% men) were included as a control. All control subjects were non-diabetic, normotensive and aged between 30-60 yr. The study was approved by the Institutional Ethics Committee of the Sri Ramachandra University, Chennai, India. A written informed consent was obtained from the study participants. The total number of cysts in the ADPKD patients was detected by using ultrasound imaging and the estimated glomerular filtration rate (eGFR) was assessed based on Modification of Diet in Renal Disease (MDRD) formula  . Further, the stage of kidney disease was determined by using eGFR and all the ADPKD patients were divided into two groups: early stages (CKD stages 1-3; eGFR 30 to 90 ml/min/1.73 m 2 and advanced (CKD stages 4; eGFR 15-29 ml/min/1.73 m 2 and 5; eGFR < 15 ml/min/1.73 m 2 stages. Genomic DNA from the peripheral blood samples was extracted by phenol chloroform extraction and ethanol precipitation protocol  .
SNP selection and genotyping: All tagging single nucleotide polymorphisms (tagSNPs) covering ACE gene from the release 2.0 Phase II data of the HapMap Project (www.hapmap.org) were selected using pairwise tagging method  . The tagSNPs were chosen according to the following criterion: r  ≥0.8 and minor allele frequency of ≥5 per cent in the Gujarati Indians in Houston (GIH) population , . A common well studied ID polymorphism was also included  . Fluorescence resonance energy transfer (FRET)-based KASPar SNP genotyping assay method (KBioscience, Herts, UK) was used to genotype the tag-SNPs on an Applied Biosystems thermocycler (ABI Prism 9700, Foster City, CA, USA) using the fluorescent probe primers (designed by K Bioscience, UK using Kraken TM software system). The fluorescent endpoint readings were taken using the 7900 SDS software (ABI Prism 7900, Foster City, CA, USA). The genotyping success rate ranged from 99.3 to 100.0 per cent. The polymerase chain reaction (PCR) and electrophoresis were used to detect the ACE gene insertion deletion polymorphism  . All homozygous deletion samples were further subjected to a second PCR ampliﬁcation with insertion-speciﬁc primers to prevent mistyping of heterozygous genotypes  .
Statistical analysis: Allele frequencies were calculated by the gene-counting method . Genotypes at each SNP were tested for Hardy-Weinberg equilibrium by using χ test with one degree of freedom. c0 hi square test was carried out to check the association between ADPKD and controls. Pairwise linkage disequilibrium (LD) measures (D' and r  and haplotype blocks were assessed using the default setting of the Haploview software  . In ADPKD patients univariate and multivariate logistic regression analysis was performed to assess the effect of genotypes and hypertension on CKD progression. The influence of different genotypes on the relationship between different CKD stages and hypertension and their interaction was examined using the Mantel-Haenszel stratified analysis. All statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, USA).
| Results|| |
ACE gene polymorphisms and ADPKD: Clinical characteristics of both controls and ADPKD patients are given in [Table 1]. The patients had significantly higher (P<0.001) creatinine and blood urea nitrogen levels and significantly (P<0.001) lower eGFR and calcium levels as compared to controls. For ACE gene GIH population yielded six tagSNPs (rs4293, rs4309, rs4311, rs4325, rs4329 and rs4362)  . The allele and genotype frequencies of the six tagSNPs along with insertion deletion polymorphisms in patients and controls are shown in t0 able II. All polymorphisms except rs4293 followed Hardy-Weinberg equilibrium. Distribution of ACE genotypes between the control and ADPKD groups was not significant [Table 2]. Analysis of LD showed a strong and significant LD between the markers by forming two LD blocks [Figure 1]. The ﬁrst LD block included rs4311 and rs4325, which were separated by 2.4 kb in introns 9 and 13, respectively. The second LD block included rs4329, I/D and rs4362, which encompassed intron 13, intron 16 and exon 21, respectively. The rs4309 SNP located in exon 8 remained outside the LD blocks. Haplotypes constructed using the SNPs located in individual LD blocks are presented in the Figure. Comparison of haplotypes between ADPKD and control groups revealed absence of any signiﬁcant association with ADPKD.
|Figure 1. Linkage disequilibrium (LD) map and haplotype analysis of ACE. Relative positions of the tagSNPs and their MAFs were shown in the upper portion. Pairwise LD measures (D and r2) were shown by the LD map. The ACE gene haplotypes distribution in control and ADPKD patients was shown in the bottom|
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|Table 1. Clinical characteristics of patients with autosomal dominant polycystic kidney disease (ADPKD) and controls |
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ACE gene polymorphisms CKD advancement: Among ADPKD patients, 49 (48%) showed advanced CKD stage with mean age of 50.73 ± 10.27 yr and 53 (52%) showed moderate progression with 43.4 ± 11.74 yr of age. Distribution of genotypes between these CKD groups revealed that only rs4362 was associated with the development of CKD stage ( P=0.046). The TT genotype significantly increased the risk of advanced CKD in ADPKD (OR=4.97; 95% CI: 1.06-32.52) [Table 3]. The univariate analysis revealed that the age, hypertension and family history of diabetes contributed to the significant advancement of CKD, but the gender had no effect on CKD advancement [Table 3]. No evidence of heterogeneity of the effect of hypertension on CKD stages was observed among different genotypes of the studied polymorphisms. This indicated the absence of confounding effect of genotype on the relationship between CKD advancement and hypertension. For rs4362 genotypes the Mantel-Haenszel (M-H) combined OR for hypertension was found to be 4.81 and 95 per cent CI is 1.26-18.4 [Table 4]. Both univariate (OR=4.97; 95% CI: 1.06-32.52) [Table 3] and multivariate analyses (OR=17.04; 95% CI: 2.38-121.95) revealed that the TT genotype of rs4362 increased the risk of CKD advancement in ADPKD after controlling age, sex, hypertension and family history of diabetes [Table 5]. The adjusted effect of hypertension on CKD advancement was found to be independent of other risk factors (OR=6.63; CI: 1.38-31.81).
|Table 3. Characteristics of patients with ADPKD and their association with chronic kidney disease (CKD) stage |
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|Table 4. Association between CKD (chronic kidney disease) stages and hypertension stratified by ACE genotypes |
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|Table 5. Adjusted effects of risk factors on chronic kidney disease (CKD) stages |
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| Discussion|| |
Analysis of tagSNPs within the ACE gene in ADPKD patients and control subjects did not show any significant association with ADPKD. Strong LD was found among all SNPs studied, covering a region of about 13.8 kb within the ACE gene. Comparison of haplotypes between ADPKD and control groups also revealed the absence of a significant association with ADPKD. The univariate analysis revealed that the age, hypertension, family history of diabetes and ACE rs4362 contributed to the advancement of CKD. The modifier effect of these factors remained even after controlling other variables in multivariate analysis, indicating that the ACE genotypes are effect modifiers of the relationship between hypertension and CKD advancement among the ADPKD patients.
Our study had some limitations. First, it was a nested study, hence there was a possibility of selection bias. Second, was the limited sample size in case group, the estimated 95 per cent CI of the effect of TT genotype on the advancement of CKD stages was found to be wider. Despite these limitations, analysis of tagSNPs of ACE gene revealed that the ACE polymorphism had a significant modifier effect on the progression of renal disease in ADPKD.
In ADPKD, hypertension is an early sign that occurs even before the reduction in glomerular filtration rate. Hence hypertension is one of the most important factors that influences the CKD progression in ADPKD ,. Inappropriate activation of RAAS has been shown to be the major cause in the pathogenesis of hypertension in individuals with ADPKD  . However, the MDRD (Modification of Diet in Renal Disease) study  demonstrated that the ACE inhibitor therapy or blood pressure control did not reduce the rate of decline in GFR of ADPKD patients. In contrast, pharmacological blockade of the RAAS using ACE inhibitors significantly reduced CKD progression  . ACE is suspected to play a key role and is the first candidate modifier gene that has been investigated in ADPKD  . An initial study of ACE gene found that the DD genotype increased the risk of developing end stage renal failure at an early age in PKD1 patients  . Subsequent studies on the influence of ACE I/D polymorphism on ADPKD progression have yielded conflicting results ,, . A comprehensive meta-analysis encompassing all relevant studies did not support the hypothesis that the DD genotype increased risk of developing end stage renal failure in patients with ADPKD  .
The synonymous marker rs4362 (C19329T) located within the 5'end of exon 23 was considered the functional candidate. It has been shown that the rs4362 marks the angiotensin-converting enzyme gene insertion-deletion variant  . A study conducted in Mexican individuals  revealed that the rs4362 polymorphisms were associated with an increased risk of hypertension and ACE serum levels. These results suggest that the rs4362 polymorphisms can modify the renal function and leads to CKD progression by altering the plasma ACE levels.
A study which examined the detailed renal functions of young ADPKD patients showed abnormal kidney function at younger age group (27 ± 5 yr) and GFR decreased but was not significantly different from that of the normal healthy controls  . Moreover, the measured eGFR in relation to age in the hypertensive children with ADPKD was an observed risk for increased renal volume and decreased renal function as compared children with normal blood pressure  . These results suggest that the renal compensation mechanism might terminate as the age increases and with the acceleration of associated risk factors.
In conclusion, our study showed that rs4362 polymorphism in ACE gene had a significant modifier effect on the progression of renal disease in patients with ADPKD.
| Acknowledgment|| |
This research was partially supported by "GATE-Young Faculty Research Grant" of the Sri Ramachandra University, Porur, Chennai.
Conflicts of Interest : None.
| References|| |
Sun Y, Zhou H, Yang BX. Drug discovery for polycystic kidney disease. Acta Pharmacol Sin
Wu G, Tian X, Nishimura S, Markowitz GS, D'Agati V, Park JH, et al.
Trans-heterozygous Pkd1 and Pkd2 mutations modify expression of polycystic kidney disease. Hum Mol Genet
Hoefele J, Mayer K, Scholz M, Klein HG. Novel PKD1 and PKD2 mutations in autosomal dominant polycystic kidney disease (ADPKD). Nephrol Dial Transplant
Devuyst O. Variable progression of autosomal dominant polycystic kidney disease: genetic and molecular counterparts. Nephrol Ther
(Suppl 2): S104-8.
Koptides M, Deltas CC. Autosomal dominant polycystic kidney disease: molecular genetics and molecular pathogenesis. Hum Genet
Fain PR, McFann KK, Taylor MR, Tison M, Johnson AM, Reed B, et al.
Modifier genes play a significant role in the phenotypic expression of PKD1
. Kidney Int
Grobe JL, Grobe CL, Beltz TG, Westphal SG, Morgan DA, Xu D, et al.
The brain renin-angiotensin system controls divergent efferent mechanisms to regulate fluid and energy balance. Cell Metab
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1
) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res
Mizuiri S, Hemmi H, Kumanomidou H, Iwamoto M, Miyagi M, Sakai K, et al.
Angiotensin-converting enzyme (ACE
) I/D genotype and renal ACE
gene expression. Kidney Int
Baboolal K, Ravine D, Daniels J, Williams N, Holmans P, Coles GA, et al.
Association of the angiotensin I converting enzyme gene deletion polymorphism with early onset of ESRF in PKD1 adult polycystic kidney disease. Kidney Int
Ravine D, Gibson RN, Walker RG, Sheffield LJ, Kincaid-Smith P, Danks DM. Evaluation of ultrasonographic diagnostic criteria for autosomal dominant polycystic kidney disease 1. Lancet
Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med
Sambrook J, Russell DW. Molecular cloning : a laboratory manual.
ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2001.
Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics
Thorisson GA, Smith AV, Krishnan L, Stein LD. The International Hap Map Project Web site. Genome Res
Shanmugam V, Sell KW, Saha BK. Mistyping ACE heterozygotes. PCR Methods Appl
Ceppellini BR, Siniscalco M, Smith CAB. The estimation of gene frequencies in a random-mating population. Ann Hum Genet
Torres VE, Grantham JJ, Chapman AB, Mrug M, Bae KT, King BF Jr, et al.
Potentially modifiable factors affecting the progression of autosomal dominant polycystic kidney disease. Clin J Am Soc Nephrol
Schrier RW, Johnson AM, McFann K, Chapman AB. The role of parental hypertension in the frequency and age of diagnosis of hypertension in offspring with autosomal-dominant polycystic kidney disease. Kidney Int
Ramanathan G, Elumalai R, Periyasamy S, Lakkakula B. Role of renin-angiotensin-aldosterone system gene polymorphisms and hypertension-induced end-stage renal disease in autosomal dominant polycystic kidney disease. Iran J Kidney Dis
Levey AS, Greene T, Beck GJ, Caggiula AW, Kusek JW, Hunsicker LG, et al.
Dietary protein restriction and the progression of chronic renal disease: what have all of the results of the MDRD study shown? Modification of diet in renal disease study group. J Am Soc Nephrol
Lattanzio MR, Weir MR. Does blockade of the renin-angiotensin-aldosterone system slow progression of all forms of kidney disease? Curr Hypertens Rep
Perez-Oller L, Torra R, Badenas C, Mila M, Darnell A. Influence of the ACE
gene polymorphism in the progression of renal failure in autosomal dominant polycystic kidney disease. Am J Kidney Dis
Lee KB, Kim UK, Lee CC. Association of the ACE
gene polymorphism with the progression of autosomal dominant polycystic kidney disease. J Korean Med Sci
Gumprecht J, Zychma MJ, Karasek D, Grzeszczak W. ACE
gene I/D polymorphism and the presence of renal failure or hypertension in autosomal dominant polycystic kidney disease (ADPKD). Nephrol Dial Transplant
Pereira TV, Nunes AC, Rudnicki M, Magistroni R, Albertazzi A, Pereira AC, et al.
Influence of ACE
I/D gene polymorphism in the progression of renal failure in autosomal dominant polycystic kidney disease: a meta-analysis. Nephrol Dial Transplant
Marciante KD, Bis JC, Rieder MJ, Reiner AP, Lumley T, Monks SA, et al.
Renin-angiotensin system haplotypes and the risk of myocardial infarction and stroke in pharmacologically treated hypertensive patients. Am J Epidemiol
Martinez-Rodriguez N, Posadas-Romero C, Villarreal-Molina T, Vallejo M, Del-Valle-Mondragon L, Ramirez-Bello J, et al
. Single nucleotide polymorphisms of the angiotensin-converting enzyme (ACE) gene are associated with essential hypertension and increased ACE enzyme levels in Mexican individuals. PLoS One
Meijer E, Rook M, Tent H, Navis G, van der Jagt EJ, de Jong PE, et al.
Early renal abnormalities in autosomal dominant polycystic kidney disease. Clin J Am Soc Nephrol
Cadnapaphornchai MA, McFann K, Strain JD, Masoumi A, Schrier RW. Prospective change in renal volume and function in children with ADPKD. Clin J Am Soc Nephrol
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]